Unit 5: Genetic Engineering

Question 1

What does Reverse Transcriptase do?

Answer 1

makes complimentary DNA from mRNA

Question 2

Describe the process of making cDNA from mRNA: (3 marks)

Answer 2

● mRNA is isolated from cells
● mRNA is mixed with FREE DNA NUCLEOTIDES and REVERSE TRANSCRIPTASE ENZYME
● Reverse Transcriptase uses mRNA as a TEMPLATE to synthesise new strand of cDNA

Question 3

What does “PALINDROMIC” mean?

Answer 3

● antiparallel BASE PAIRS
● which are read the same FORWARD AND BACK

(Recognition Sequence)

Question 4

Define “CODON”:

Answer 4

● 3 bases on mRNA

● which code for an AMINO ACID

Question 5

What is the role of DNA Polymerase?

Answer 5

JOINS nucleotides together to form (mRNA)

Question 6

Name and describe the role of the enzyme which recognises recognition sequences: (4 marks)

Answer 6

Name = Restriction Endonucleas Enzyme
● DNA sample is INCUBATED with SPECIFIC restriction endonuclease
● this CUTS the DNA fragment out via HYDROLYSIS
● sometimes cut leaves STICKY ENDS

Question 7

What are STICKY ends and what’s their role? (2 marks)

Answer 7

(small tails of UNPAIRED BASES at each end of DNA fragment)

● they can ANNEAL the DNA fragment to another piece of DNA that has sticky ends with COMPLIMENTARY SEQUENCES

Question 8

What does PCR stand for and describe the process: (7 marks)

Answer 8

[ POLYMERASE CHAIN REACTION ]
● BACTERIAL DNA mixed with free nucleotides, primers and DNA POLYMERASE
● Mixture heated to 95○C to BREAK H BONDS
● Mixture cooled to between 50-65 ○C to allow PRIMERS to anneal to DNA
● primers anneal they have complimentary sequence to the sequence at the START of DNA fragment
● Mixture heated to 72 ○C and DNA POLYMERASE lines up FREE NUCLEOTIDES along each template strand, producing new strands of DNA
● Cycle repeated to produce lots of copies

Question 9

Define in Vivo cloning:

Answer 9

where gene copies are made WITHIN a LIVING organism

Question 10

Define In Vitro Cloning:

Answer 10

where gene copies are made OUTSIDE of a living organism using PCR

Question 11

Define “Vector”:

Answer 11

something that’s used to TRANSFER DNA INTO A CELL

Question 12

What two things can act as VECTORS?

Answer 12

● Plasmids

● BACTERIOPHAGES (viruses that infect bacteria)

Question 13

Why is the SAME RESTRICTION ENDONUCLEAS ENZYME used to cut open Vector DNA and isolate DNA fragment?

Answer 13

so the STICKY ENDS of the VECTOR is COMPLIMENTARY to the STICKY ENDS OF DNA FRAGMENT

Question 14

Define “Ligation”:

Answer 14

● process where DNA LIGASE joins the STICKY ENDS of DNA fragment to the STICKY ENDS of the VECTOR DNA
● to form RECOMBINANT DNA

Question 15

How can a PLASMID vector transfer gene into HOST cells?

Answer 15

1) host bacterial cells placed into ice-cold CALCIUM CHLORIDE to make CELL WALLS more PERMEABLE
2) Plasmids are added and mixture is HEAT-SHOCKED (eated to 42 ○C for 1-2 mins) which encourages cells to take in plasmid

Question 16

How can a BACTERIOPHAGE vector transfer gene into HOST cells?

Answer 16

Injects the phage DNA (with TARGET GENE IN IT) Into the host bacterium and this integrates into bacterial DNA